Immunopotentiating and infection protective agent and production thereof

ABSTRACT

An immunopotentiating and infection protective agent comprising riboflavin and/or a riboflavin derivative, which is safe for the human, animals or the like, an immunopotentiating and infection protective agent comprising riboflavin and/or a riboflavin derivative and (1) proline and/or glutamine, (2) an antibiotic, or (3) a water-soluble polymer or lecithin, which is safe for the human, animals or the like, and a process for the production thereof.

This application is a divisional of now abandoned application, Ser. No.08/204,333, filed Mar. 14, 1994; which is the U.S. national stageapplication of PCT/JP92/01146, filed Sep. 9, 1992.

INDUSTRIALLY APPLICABLE FIELD

The present invention relates to medicines for potentiating the immunefunction of the human, animals or the like so as to protect them frominfection, and a process for the production thereof.

PRIOR ART AND BACKGROUND OF THE INVENTION

Owing to the recent advancement of immunology, various disorders orinfectious diseases of the human and animals have come to be consideredto be caused by reduction or failure in immune function.

In the case of the human, for example, immune function is reduced orbrought to failure by bronchial asthma, allergic diseases, articularrheumatism, autoimmune diseases, nutritional disorders, surgicaloperations, advanced age, pregnancy, etc. in many cases, so thatinfectious diseases such as respiratory infection, sepsis and urinarytract infection are developed at the same time. Various kinds ofantibiotics have heretofore been administered against these disordersand infectious diseases.

In livestock and marine products industries on the other hand,large-scale operation or overcrowded breeding has been conducted inorder to rear livestock, poultry or cultured fishes with highefficiency. The massive administration of antibiotics have been adoptedin such breeding.

Under such circumstances, Japanese Patent Application Laid-Open (KOKAI)No. 286923/1987 discloses the fact that proline(pyrrolidine-2-carboxylic acid), which is a sort of amino acid producedby the decomposition of a protein, has an immune-enhancing action.Further, Japanese Patent Publication No. 38985/1988 discloses the factthat glutamine, which is a derivative of proline, has animmune-enhancing action.

Riboflavin useful in the practice of the present invention isphosphorylated in vivo to form twenty-odd flavoenzymes such as aminoacid oxidases and xanthine oxidases as coenzymes (for example, flavinmononucleotide and flavin adenine dinucleotide), so that it participatesin the oxidation-reduction mechanism of organism and plays an importantpart in metabolism of carbohydrates, lipids, proteins, etc.

In the case of the human, it has been known that the lack of riboflavinleads to cheilitis, acute chronic eczema, solar eczema, seborrheiceczema, conjunctivitis, angular cheilitis, glossitis, pellagra or thelike. Accordingly, riboflavin is used for preventing and treating thesedeficiency diseases of vitamine B₂.

Derivatives of riboflavin have been known to have substantially the samepharmacological action as that of riboflavin and to be applied to thesame disorders or diseases.

THEMES TO BE SOLVED BY THE INVENTION

When a specific antibiotic is used continuously, its resistant bacteriagenerates and the efficacy of the antibiotic is lowered. Further, thereis also a problem of nosocomial infection recently highlighted.Therefore, there is a demand for the development of a prophylactic andtherapeutic drug which permits reduction in the amount of antibiotics tobe used and can enhance immune function.

In the overcrowded breeding in the livestock and marine productsindustries on the other hand, there is a problem that various infectiousdiseases often develop due to stress and immunodeficiency in juvenileyears. The massive administration of antibiotics as its countermeasureis accompanied this time by problems of retention of the antibiotics andincrease of resistant bacteria.

In view of the above-described problems involved in antibiotics, thepresent inventors have carried out an extensive investigation for a longtime with a view toward developing a infection protective agent safe forthe human or animals. As a result, it has been found that riboflavinand/or riboflavin derivatives have an action to potentiate immunefunction, and also that water-soluble polymers and the like have anaction to enhance and sustain the immune-function-potentiating action ofriboflavin and/or the riboflavin derivatives, leading to completion ofthe present invention.

MEANS FOR SOLVING THE THEMES

The present invention relates to an immunopotentiating and infectionprotective agent comprising riboflavin and/or a riboflavin derivative.

As described above, proline and glutamine have an action to potentiateimmune function. However, it has been unexpectedly found that thecombined use of riboflavin and/or the riboflavin derivative with prolineand/or glutamine according to the present invention synergisticallyenhances the action to potentiate immune function. Therefore, thepresent invention relates to an immunopotentiating and infectionprotective agent comprising riboflavin and/or a riboflavin derivativeand proline and/or glutamine.

It has been unexpectedly found that the combined use of riboflavinand/or a riboflavin derivative and an antibiotic develops a so-calledsynergism over those achieved by their single use. As a result, suchcombined use gives an important effect that the amount of the antibioticto be used is decreased to a significant extent. Thus, the presentinvention relates to an immunopotentiating and infection protectiveagent comprising riboflavin and/or a riboflavin derivative and anantibiotic.

Further, it has been unexpectedly found that the combined use ofriboflavin and/or a riboflavin derivative and a water-soluble polymer orlecithin enhance the infection protective effect of riboflavin and/orthe riboflavin derivative. Therefore, the present invention relates toan immunopotentiating and infection protective agent comprisingriboflavin and/or a riboflavin derivative and a water-soluble polymer orlecithin.

Further, it has been unexpectedly found that the combined use ofriboflavin and/or a riboflavin derivative and a vaccine exhibits aso-called synergism over the immunopotentiating and infection protectiveeffects achieved by their single use. Thus, the present inventionrelates to a vaccine preparation comprising riboflavin and/or ariboflavin derivative and a vaccine.

The present invention is also concerned with a process for theproduction of an immunopotentiating and infection protective agentcomprising riboflavin and/or a riboflavin derivative and a water-solublepolymer or lecithin.

The immunopotentiating and infection protective agent comprisingriboflavin and/or a riboflavin derivative and lecithin, or theimmunopotentiating and infection protective agent comprising riboflavinand/or a riboflavin derivative and a water-soluble polymer can not beprepared by simple mixing because parts of riboflavin derivatives andlecithins are hard to dissolve in water. Such an agent can be preparedby emulsify the riboflavin derivative or lecithin. Therefore, thepresent invention is also related to a process for the production of animmunopotentiating and infection protective agent, which comprisesemulsifying lecithin and riboflavin and/or a riboflavin derivative in asolvent, or emulsifying riboflavin and/or a riboflavin derivative and awater-soluble polymer in a solvent.

It is an object of the present invention to provide an agent which canpotentiate immune function and is safe for the human or animals withoutthe above-described drawbacks involved in the administration ofantibiotics, thereby permitting the protection of organism frominfection, and a process for the production thereof.

The term "immunopotentiating" as used herein means enhancing immunefunction in the human, animals, for example, fishes, or the like.

Therefore, since the immunopotentiating and infection protective agentsaccording to the present invention are useful as agents for enhancingthe immune function of the human, animals or the like so as to preventand treat various disorders and infectious diseases, no particularlimitation is imposed on their cases to be applied. In the case of thehuman, they are applied to, for example, cancers, organtransplantations, leukopenia, articular rheumatism, autoimmune diseases,bronchial asthma, nutritional disorders, surgical operations, agediseases and various infectious diseases such as respiratory infection,sepsis and urinary tract infection.

In the case of the animals, they are applied to, for example, thediarrhea, epidemic pneumonia, atrophic rhinitis, infectiousgastroenteritis and the like of swine, the pneumonia and Marek's diseaseof domestic fowl, the diarrhea, pneumonia and udder inflammation ofbovine, the AIDS of pets and the leukemia of cats.

Further, no particular limitation is imposed on infectious diseases ofcultured fishes, to which the immunopotentiating and infectionprotective agents according to the present invention are applied.However, they are used widely for bacterial infections such asstreptococcosis and pseudotuberculosis, virus infections, and the like.

In the present invention, riboflavin and the riboflavin derivative maybe used either singly or in combination. Examples of the riboflavinderivative include flavin mononucleotide, flavin adenine nucleotide andpharmacologically permissible salts of riboflavin (for example, sodiumriboflavin phosphate, the monodiethanolamine salt of riboflavinphosphate, etc.).

No particular limitation is imposed on the amount of riboflavin and/orthe riboflavin derivative to be used in the present invention because itvaries according to the species of animal to be applied, and the like.In general, its dose falls within a range of 0.1-500 mg/kg of weight,preferably 1-100 mg/kg of weight.

No particular limitation is imposed on the compounding ratio ofriboflavin and/or the riboflavin derivative to proline and/or glutaminein the present invention. However, the compounding ratio of prolineand/or glutamine is generally 0.1-10 parts by weight, preferably 0.5-5parts by weight based on 1 part by weight of riboflavin and/or theriboflavin derivative.

In the present invention, proline and glutamine may be incorporatedsingly into riboflavin and/or the riboflavin derivative. Alternatively,a mixture of both proline and glutamine may be incorporated intoriboflavin and/or the riboflavin derivative.

Further, no particular limitation is imposed on the kind of theantibiotic used in combination with riboflavin and/or the riboflavinderivative. However, amoxicillin, tetracycline, oxycycline hydrochloridemay be mentioned by way of example.

Amoxicillin is a penicillin antibiotic, has an antibacterial actionowing to the inhibition of cell wall synthesis, and is applied tovarious infectious diseases caused by Escherichia coli, Haemophilusinfluenzae, haemolytic streptococcus, staphylococcus and the like, whichare sensitive to amoxicillin.

Further, no particular limitation is imposed on the compounding ratio ofriboflavin and/or the riboflavin derivative to the antibiotic in thepresent invention. However, the compounding ratio of the antibiotic isgenerally 0.01-1 part by weight, preferably 0.05-0.5 part by weightbased on 1 part by weight of riboflavin and/or the riboflavinderivative.

Further, no particular limitation is imposed on the compounding ratio ofriboflavin and/or the riboflavin derivative to the water-soluble polymeror lecithin in the present invention. However, the compounding ration ofthe water-soluble polymer or lecithin is generally 0.01-100 parts byweight, preferably 0.05-50 parts by weight, more preferably 0.1-10 partsby weight based on 1 part by weight of riboflavin and/or the riboflavinderivative.

No particular limitation is imposed on the water-soluble polymer.However, preferred water-soluble polymers include polyvinyl pyrrolidone,sodium carboxymethyl cellulose, methyl cellulose, hydroxypropylcellulose, hydroxypropylmethyl cellulose, sodium chondroitin sulfate,polyethylene-hardened castor oil, polyoxysorbitan fatty acid esters andpolyvinyl alcohol. These polymers may be used singly or in anycombination thereof.

No particular limitation is imposed on the lecithin. However, yolklecithin, soybean lecithin and hydrogenated lecithins thereof may bementioned and used in a single form or in any combination thereof.

Further, no particular limitation is imposed on the kind and compoundingratio of the vaccine to be used in combination with riboflavin and/orthe riboflavin derivative in the present invention because they variesaccording to the species to be applied, such as the human or animals,e.g., fishes. However, examples of such a vaccine include various kindsof vaccines such as a chicken mycoplasma vaccine, chicken infectiouscoryza type A.C inactivated vaccine, swine Bordetella inactivatedvaccine and swine Haemophilus (Actinobacillus) inactivated vaccine inthe case of the animals.

No particular limitation is imposed on the form of theimmunopotentiating and infection protective agent according to thepresent invention when it is administered to the human or an animal.However, it may be formed into an injection, granules, powder, tablets,or the like.

When the immunopotentiating and infection protective agent according tothe present invention is prepared, various kinds of additives may beincorporated according to the form prepared. For example, an excipient,colorant, lubricant, binder, coating and the like may be incorporatedwhen prepared in the form of a solid or powder.

When low-solubility substances are prepared in injections, a dissolutionaid such as a surfactant is often used. In the present invention, asurfactant such as polyoxyethylene-hardened castor oil, or the like isalso used. These substances are added on the basis of an unexpectedfinding that they can enhance the immunopotentiating and infectionprotective action of riboflavin and/or the riboflavin derivative, andhence do not have a mere effect as a dissolution aid.

The immunopotentiating and infection protective agent according to thepresent invention, which comprises riboflavin and/or the riboflavinderivative, or proline and/or glutamine in addition to riboflavin and/orthe riboflavin derivative, may be added to food so as to use it as afood specifically intended for the prevention of individual diseases ordisorders and having a biological control function, i.e., a so-calledfunctional food.

Further, since the immunopotentiating and infection protective agentaccording to the present invention is free of the influence of resistantbacteria and the problem of retention, which are recognized inantibiotics, it may be used for livestock such as swine, domestic fowl,bovine, equine and ovine, fishes, pets (dogs, cats, birds), and the likeas a safe feed having a biophylatic control function, i.e., a functionalfeed.

The immunopotentiating and infection protective agent according to thepresent invention, which comprises riboflavin and/or the riboflavinderivative, or an antibiotic in addition to riboflavin and/or theriboflavin derivative, is administered in the form of intramuscularinjection, intravenous injection, subcutaneous injection or oraladministration when given to the human or animals.

FUNCTION

The present inventors do not completely elucidate the mechanism ofintravital action in which the riboflavin derivatives potentiate immunefunction. However, it has been recognized that the riboflavinderivatives activate phagocytes, for example, macrophages, in leukocytesand neutrophiles. In addition, it has also been found that the number ofleukocytes (in particular, the number of neutrophiles, and the like) isincreased.

EXAMPLES

The present invention will hereinafter be described specifically by thefollowing examples. In the following examples, the description on thedoses of substances to be used, for example, "10 mg/kg i.m." means thatintramuscular injection was conducted in a proportion of 10 mg per kg ofweight. Further, the designations of "*" and "**" as will be used in thecolumn of x² -test in Tables 1 to 7 mean p<0.05 and p<0.01,respectively.

Example 1

Riboflavin in proportions of 10, 30 and 100 mg/kg and physiologicalsaline as a control were intramuscularly injected into each 10 SLC:ICRmale mice (aged 5-6 weeks, weight: 25-30 g). After 24 hours, clinicallyderived Escherichia coli (2.6×10⁷ CFU/mouse, 0.2 ml) was subcutaneouslyinoculated into the mice in each group to determine the survival ratefrom the viable count on the 7th day from the infection, thereby findingthe significance to the control. The results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Sample              Survival rate %                                                                          x.sup.2 -Test                                  ______________________________________                                        Control (physiological saline, i.m.)                                                              10                                                        Riboflavin, 10 mg/kg i.m.                                                                         20                                                        Riboflavin, 30 mg/kg i.m.                                                                         50                                                        Riboflavin, 100 mg/kg i.m.                                                                        90         **                                             ______________________________________                                    

As shown in Table 1, the effect of riboflavin increases in dependence onthe doses. It is therefore apparent that riboflavin has an infectionprotective effect. The effect of riboflavin is powerful as demonstratedby the survival rates of 50% and 90% in doses of 30 mg/kg and 100 mg/kg,respectively.

Example 2

Glutamine, proline and riboflavin, and a control (physiological saline)were intramuscularly injected into each 10 SLC:ICR male mice (aged 5-6weeks, weight: 22-30 g) either singly or in combination with each otheras shown in Table 2. After 24 hours, clinically derived Escherichia coli(2.6×10⁷ CFU/mouse, 0.2 ml) was inoculated into the mice in each groupto determine the survival rate from the viable count on the 7th day fromthe infection.

With respect to sole glutamine, proline or riboflavin and theircombinations with each other, the significance was found to the control.The results are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Sample              Survival rate %                                                                          x.sup.2 -Test                                  ______________________________________                                        Control (physiological saline, i.m.)                                                              0                                                         Glutamine, 100 mg/kg i.m.                                                                         30                                                        Proline, 100 mg/kg i.m.                                                                           40         *                                              Glutamine, 100 mg/kg;                                                                             50         *                                              Proline, 100 mg/kg i.m.                                                       Riboflavin, 10 mg/kg i.m.                                                                         20                                                        Riboflavin, 30 mg/kg i.m.                                                                         50         *                                              Riboflavin, 100 mg/kg i.m.                                                                        90         **                                             Glutamine, 100 mg/kg;                                                                             100        **                                             Proline, 100 mg/kg;                                                           Riboflavin, 10 mg/kg i.m.                                                     ______________________________________                                    

Further, with respect to the combination of glutamine, proline andriboflavin, the significance was found to the combination of glutamineand proline. The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Sample            Survival rate %                                                                          x.sup.2 -Test                                    ______________________________________                                        Glutamine, 100 mg/kg;                                                                           50                                                          Proline, 100 mg/kg i.m.                                                       Glutamine, 100 mg/kg;                                                                           100        *                                                Proline, 100 mg/kg;                                                           Riboflavin, 10 mg/kg i.m.                                                     ______________________________________                                    

As shown in Table 2, the survival rate owing to proline in a dose of 100mg/kg is 40% and proline is hence significant compared with the control.This indicates that proline has an infection protective effect. Thesurvival rates owing to riboflavin in doses of 30 mg/kg and 100 mg/kgare 50% and 90%, respectively. It is understood that riboflavin exhibitsa more powerful infection protective effect in dependence on its doseseven when compared with proline.

It was confirmed from Table 2 that the combination of glutamine, prolineand riboflavin has an effect more than the additive effect as the sum ofeffects achieved by using the respective components singly, i.e., asynergism.

In addition, it is also understood from Table 3 that the combination ofglutamine, proline and riboflavin exhibits an infection protectiveeffect as extremely powerful as 100% in survival rate. When comparedwith the additive effect of the effect in the combination of glutamineand proline and the effect in the single use of riboflavin, it wasconfirmed that the combination of the three components has a clearlysignificant synergism.

Example 3

Sodium riboflavin phosphate in proportions of 10, 30, 100 and 300 mg/kgand physiological saline as a control were intramuscularly injected intoeach 10 SLC:ICR male mice (aged 5-6 weeks, weight: 25-30 g). After 24hours, clinically derived Escherichia coli (2.6×10⁷ CFU/mouse, 0.2 ml)was subcutaneously inoculated into the mice in each group to determinethe survival rate from the viable count on the 7th day from theinfection, thereby finding the significance to the control. The resultsare shown in Table 4.

                  TABLE 4                                                         ______________________________________                                        Sample              Survival rate %                                                                          x.sup.2 -Test                                  ______________________________________                                        Control (physiological saline, i.m.)                                                              0                                                         Sodium riboflavin phosphate,                                                                      10                                                        10 mg/kg i.m.                                                                 Sodium riboflavin phosphate,                                                                      40         *                                              30 mg/kg i.m.                                                                 Sodium riboflavin phosphate,                                                                      60         **                                             100 mg/kg i.m.                                                                Sodium riboflavin phosphate,                                                                      100        **                                             300 mg/kg i.m.                                                                ______________________________________                                    

As shown in Table 4, the effect of sodium riboflavin phosphate increasesin dependence on the doses, i.e., 10, 30, 100 and 300 mg/kg. Inparticular, it was confirmed that the use of sodium riboflavin phosphatein a proportion of 300 mg/kg exhibits an extremely powerful infectionprotective effect.

Example 4

Sodium riboflavin phosphate and amoxicillin (AMPC) in proportions of 10mg/kg and 0.39 mg/kg, respectively, were intramuscularly injected intoeach 10 SLC:ICR male mice (aged 5-6 weeks, weight: 25-30 g) eithersingly or in combination with each other 24 hours before infection and30 minutes after infection. Clinically derived Escherichia coli (2.6×10⁷CFU/mouse, 0.2 ml) was subcutaneously inoculated into the mice in eachgroup to determine the survival rate from the viable count on the 7thday from the infection. The results are shown in Table 5.

                  TABLE 5                                                         ______________________________________                                        Sample              Survival rate %                                                                          x.sup.2 -Test                                  ______________________________________                                        Control (physiological saline, i.m.)                                                              0                                                         Amoxicillin, 0.39 mg/kg i.m.                                                                      60         **                                             Sodium riboflavin phosphate,                                                                      10                                                        10 mg/kg i.m.                                                                 Amoxicillin, 0.39 mg/kg;                                                                          100        **                                             Sodium riboflavin phosphate,                                                  10 mg/kg i.m.                                                                 ______________________________________                                    

As shown in Table 5, it was conformed that the combination ofamoxicillin and sodium riboflavin phosphate has an effect more than theadditive effect as the sum of effects achieved by using the respectivecomponents singly, i.e., a significant synergism.

Example 5

flavin mononucleotide (FMN) and riboflavin in proportions of 100 mg/kg,and polyvinyl pyrrolidone (PVP-K30), sodium carboxymethyl cellulose (CMCNa), purified soybean lecithin, yolk lecithin, polyoxyethylene (60)ether (HCO-60), polyoxyethylene (20) sorbitan monooleate (Tween-80) anda control (physiological saline) were intramuscularly injected into each10 SLC:ICR male mice (aged 5-6 weeks, weight: 25-30 g) in combinationwith each other as shown in the following Table 6. After 3 days,clinically derived Escherichia coli (2.6×10⁷ CFU/mouse, 0.2 ml) wassubcutaneously inoculated into the mice in each group to determine thesurvival rate from the viable count on the 7th day from the infection,thereby finding the significance to the control. The results are shownin Table 6.

                  TABLE 6                                                         ______________________________________                                        Sample                Survival rate %                                                                          x.sup.2 -Test                                ______________________________________                                        Control (physiological saline, i.m.)                                                                0                                                       FMN, 100 mg/kg i.m.   30                                                      FMN, 100 mg/kg; PVP-K30, 300 mg/kg i.m.                                                             40         *                                            FMN, 100 mg/kg; CMC Na, 30 mg/kg i.m.                                                               50         *                                            FMN, 100 mg/kg;       70         **                                           Purified soybean lecithin, 200 mg/kg i.m.                                     FMN, 100 mg/kg;       90         **                                           Yolk lecithin, 100 mg/kg i.m.                                                 FMN, 100 mg/kg i.m.; HCO-60 10 mg/kg i.m.                                                           30                                                      Riboflavin, 100 mg/kg i.m.                                                                          40         *                                            Riboflavin, 100 mg/kg;                                                                              90         **                                           PVP-K30, 300 mg/kg i.m.                                                       Riboflavin, 100 mg/kg;                                                                              80         **                                           CMC Na, 30 mg/kg i.m.                                                         Riboflavin, 100 mg/kg;                                                                              90         **                                           Purified soybean lecithin, 200 mg/kg i.m.                                     Riboflavin, 100 mg/kg;                                                                              100        **                                           Yolk lecithin, 100 mg/kg i.m.                                                 Riboflavin, 100 mg/kg;                                                                              50         *                                            HCO-60 10 mg/kg i.m.                                                          Riboflavin, 100 mg/kg;                                                                              70         **                                           Tween-80, 10 mg/kg i.m.                                                       ______________________________________                                    

As shown in Table 6, it was confirmed that the various water-solublepolymers such as polyvinyl pyrrolidone (PVP-K30), sodium carboxymethylcellulose (CMC Na), polyoxyethylene (60) ether (HCO-60) andpolyoxyethylene (20) sorbitan monooleate (Tween-80), and lecithins suchas purified soybean lecithin and yolk lecithin enhance and sustain theinfection protective effect of FMN and riboflavin.

Example 6

Riboflavin or sodium riboflavin phosphate and yolk lecithin were usedeither singly or in combination with each other as shown in Table 7 todilute them with a 20-fold phosphate buffer. Portions of the resultingdilute solutions were mixed with commercially-available Actinobacilluspleueopneumoniae inactivated vaccine to produce vaccine preparations.The thus-produced vaccine preparations, the residual dilute solutionsand a phosphate buffer as a control in amounts of 0.5 ml wereintraperitoneally administered into each 20 SLC:ICR male mice (aged 3weeks, weight: 12-15 g). Upon elapsed time of 14 days after theadministration, 0.5 ml of Actinobacillus pleueopneumoniae (3×10⁸CFU/mouse) was intraperitoneally inoculated into the mice in each groupto determine the survival rate after 7 days. The results are shown inTable 7. This experiment was carried out in accordance with the methodof national certification of vaccine.

                  TABLE 7                                                         ______________________________________                                        Sample              Survival rate %                                                                           x.sup.2 -Test                                 ______________________________________                                        Control (phosphate buffer, i.p.)                                                                  10                                                        Vaccine, i.p.       40                                                        Riboflavin, 100 mg/kg i.p.                                                                        25                                                        Riboflavin, 100 mg/kg; vaccine, i.p.                                                              80          **                                            Riboflavin, 100 mg/kg;                                                                            95          **                                            Yolk lecithin, 100 mg/kg;                                                     Vaccine, i.p.                                                                 Sodium riboflavin phosphate,                                                                      5                                                         100 mg/kg i.p.                                                                Sodium riboflavin phosphate,                                                                      50                                                        100 mg/kg;                                                                    Vaccine, i.p.                                                                 Sodium riboflavin phosphate,                                                                      60          **                                            100 mg/kg;                                                                    Yolk lecithin, 100 mg/kg; Vaccine, i.p.                                       ______________________________________                                    

As shown in Table 7, it was confirmed that the combination of riboflavinor sodium riboflavin phosphate and the vaccine has an effect more thanthe additive effect as the sum of infection protective effects achievedby using the respective components singly, i.e., a significantsynergism. This synergism means the enhanced infection protective effectof the vaccine, i.e., is nothing but the enhancement effect on thevaccine.

Example 7

Five grams of flavin mononucleotide (FMN), 5 g of D-sorbitol, 0.04 g ofdisodium phosphate, 0.04 g of monosodium phosphate and 15 g of polyvinylpyrrolidone (PVP-K30) were dissolved in water for injection into 100 mlof a solution. The resulting solution was poured in parts into 5-mlampules and sterilized with steam, thereby preparing immunopotentiatingand infection protective agents.

Example 8

Immunopotentiating and infection protective agents were prepared in thesame manner as in Example 7 except that 3 g of hydroxypropyl cellulose(HPC) was used in place of 15 g of PVP-K30 in Example 7.

Example 9

Immunopotentiating and infection protective agents were prepared in thesame manner as in Example 7 except that 2 g of hydroxypropylmethylcellulose (HPMC) was used in place of 15 g of PVP-K30 in Example 7.

Example 10

Immunopotentiating and infection protective agents were prepared in thesame manner as in Example 7 except that 20 g of sodium chondroitinsulfate was used in place of 15 g of PVP-K30 in Example 7.

Example 11

After 10 g of yolk lecithin was dispersed in an ultrasonic emulsifier, 5g of D-sorbitol, 0.03 g of disodium phosphate, 0.02 g of monosodiumphosphate and 3 g of FMN were dissolved in the resulting dispersion,followed by dissolution of the resulting solution in water for injectioninto 100 ml of another solution. The thus-obtained solution was pouredin parts into 5-ml ampules and sterilized with steam, thereby preparingimmunopotentiating and infection protective agents.

Example 12

Immunopotentiating and infection protective agents were prepared in thesame manner as in Example 11 except that 10 g of partially hydrogenatedsoybean lecithin was used in place of 10 g of yolk lecithin in Example11 and the amount of FMN was changed to 4 g.

Example 13

Five grams of microcrystalline riboflavin were suspended in water forinjection, which contained 5 g of D-sorbitol, 1 g of sodiumcarboxymethyl cellulose (CMC Na), 0.04 g of disodium phosphate and 0.04g of monosodium phosphate, into 100 ml of a suspension. This suspensionwas dispersed in an ultrasonic emulsifier. The resulting dispersion waspoured in parts into 5-ml ampules and sterilized with steam, therebypreparing immunopotentiating and infection protective agents.

Example 14

Immunopotentiating and infection protective agents were prepared in thesame manner as in Example 13 except that 3 g of HPMC was used in placeof 1 g of CMC-Na in Example 13

Example 15

Immunopotentiating and infection protective agents were prepared in thesame manner as in Example 13 except that 3 g of polyvinyl alcohol wasused in place of 1 g of CMC-Na in Example 13.

Example 16

After 10 g of partially hydrogenated yolk lecithin and 5 g of D-sorbitolwere dispersed in an ultrasonic emulsifier, 0.03 g of disodiumphosphate, 0.02 g of monosodium phosphate and 3 g of FMN were dissolvedin the resulting dispersion. The resulting solution was added with 5 gof riboflavin to suspend it, followed by dissolution of the resultingsuspension in water for injection into 100 ml of another solution. Thethus-obtained solution was poured in parts into 5-ml ampules andsterilized with steam, thereby preparing immunopotentiating andinfection protective agents.

The immunopotentiating and infection protective agents according to thepresent invention were prepared in accordance with the processes for theproduction described in Examples 7 to 16.

EFFECT OF THE INVENTION

From the above Examples, the immunopotentiating and infection protectiveagents and vaccine preparations have an excellentimmune-function-potentiating action. Therefore, they are useful asprophylactic and therapeutic drugs for various disorders and infectiousdiseases.

We claim:
 1. A method for treatment of diseases selected from the groupconsisting of: in humans, leukopenia, autoimmune diseases, sepsis andurinary tract infection; in swine, diarrhea, epidemic pneumonia,atrophic rhinitis and infectious gastroenteritis; in domestic fowl,pneumonia and Marek's disease; in bovines, diarrhea, pneumonia and udderinflammation; and in cats, leukemia, which comprises administering to ahuman or animal patient selected from the group consisting of swine,domestic fowl, bovines and cats, in need of such treatment a compositionconsisting of riboflavin and/or a riboflavin derivative.
 2. The methodaccording to claim 1, wherein the composition is administered in anamount of 0.5-500 mg/kg.